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Abstract
Efficient preparation and labeling of human induced pluripotent stem (iPS) cells is a great challenge in stem cell research and development. With the aim of investigating the feasibility of using nanotechnology to enhance the preparation efficiency of iPS cells and to label iPS cells for long-term tracing and imaging, in this paper, four transcription factor genes, ie, Oct4, Sox2, LIN28, and Nanog, and packaging plasmids such as PSPAX2 and PMD2.G were cotransfected into 293T cells using Generation 5.0 polyamidoamine dendrimer-modified magnetic nanoparticles (dMNPs) as a delivery system. The resultant supernatant liquids were incubated with human fibroblast cells at 37°C for 21 days, then the embryonic stem (ES) cell-like clones were screened, cultured, and identified. Finally, the prepared iPS cells were labeled with fluorescent magnetic nanoparticles (FMNPs). The results showed that dMNPs can efficiently deliver all vectors into 293T cells. The resultant lentiviruses’ titers were 10-fold more than those based on Lipofectamine™ 2000. Reverse transcription polymerase chain reaction analysis showed that four genes (Oct4, Sox2, LIN28, and Nanog) exhibited different expressions in iPS cells. Immunostaining analysis showed that specific surface markers of ES cells such as SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81 were positive in iPS cells, and the terotomas were formed in NOD-SCID mice that were implanted with iPS cells. Red fluorescent signals could be observed in iPS cells labeled with FMNPs by fluorescent microscopy, and the magnetic signals were detected in labeled iPS cells by magnetic resonance imaging. In conclusion, human iPS cells can be efficiently generated using polyamidoamine dMNPs and lentivirus and labeled with FMNPs for long-term observation and tracking, which has great potential application in the research and development of stem cells in the near future.
Supplemental information
Figure S1 The digestion maps of plasmid DNA. a) Single-site digestion map of plasmids DNA. b) Double-site digestion map of plasmid DNA.
Figure S2 Characterizations of dendrimer-modified magnetic nanoparticles (dMNPs). A) Transmission electron microscope image of dMNPs. B) The Fourier transform infrared spectra of dMNPs: a) MNPs, b) dMNPs.
Figure S3 Characterizations of fluorescent magnetic nanoparticles (FMNPs). A) The transmission electron microscope image of FMNPs. B) The fluorescent image of FMNPs with/without external magnetic field. C) The field-dependent magnetization curve of FMNPs at room temperature. D) The photoluminescence spectra of FMNPs.
Acknowledgements
This work is supported by the National Key Basic Research Program (973 Project) (2010CB933901), National 863 Hi-tech Project (2007AA022004), Important National Science and Technology Specific Project (2009ZX10004-311), National Natural Scientific Fund (No. 20771075 and No. 20803040), Special Project for Nano-technology from Shanghai (No. 1052nm04100), New Century Excellent Talent of Ministry of Education of China (NCET-08-0350, No. 20070248050), Shanghai Science and Technology Fund (10XD1406100), and Pu Jiang Project (09PJ1407300).
Disclosure
The authors report no conflicts of interest in this work.